Acute and chronic thrombotic states are major causes of morbidity and mortality in many industrialized countries. Occlusion of blood vessels by thrombus or fibrin clots plays an integral role in both heart and pulmonary disease. Plasmin is a protease with relative broad substrate specificity. In blood, plasmin cleaves fibrin, thus, resulting in lysis of the fibrin clot. Effective therapies must also be capable of lysing these fibrin clots.
One approach to the treatment of an established thrombosis is the pharmacological dissolution of the blood clot via intravenous infusion of plasminogen activators. Plasminogen activators are proteolytic enzymes possessing specific proteolytic activity towards the blood plasma protein plasminogen. There are several plasminogen activators (PA) including, but not limited to, tissue-type PA (t-PA), urokinase PA (u-PA: including the proenzyme form referred to as prourokinase, or single chain urokinase PA (scu-PA); high molecular weight two chain uPA and low molecular weight uPA), and streptokinase, which are capable of converting inactive zymogen plasminogen to the active enzyme, plasmin, by specific cleavage of plasminogen. Plasmin digests fibrin to soluble degradation products, thus, eliminating the clot. Plasminogen activators have been demonstrated to cause marked decreases in plasminogen and fibrinogen concentrations while increasing thrombin time, activated partial thromboplastin time and prothrombin time.
All of the plasminogen activators provided are presently in worldwide clinical trials as fibrinolytic agents. Endogenous tPA and scuPA demonstrate selectivity toward fibrin, thus, few systemic effects from this treatment have been predicted. However, provision of these activators in pharmacological amounts has been found to overwhelm normal control mechanisms such as plasminogen activator inhibitors. The major problems associated with the efficacy of these agents are extremely fast blood clearance, rapid re-occlusion and hemorrhaging in tissues such as the brain. Accordingly, new therapies and delivery systems are being investigated.
The objective for the ideal fibrinolytic agent has been defined as the rapid achievement of vascular patency in all patients without early re-occlusion or bleeding. Primary aims for new therapies include increasing plasma clearance half-life, decreasing inhibitor interactions, and improving fibrin affinity and localization of the action of plasminogen activator(s) in definitive sites of the vasculature. Sites of fibrin clot formation, sites of deposition or embolization of fibrin clots and sites with high probability of vascular occlusion by fibrin clots represent such definitive sites of the vasculature. Mitchel et al. Circulation (1995) 91(3):785-783.
Targeted delivery of these activators to the site of clot formation has been performed in a number of different manners. Plasminogen activators have been artificially conjugated with monoclonal antibodies to fibrin providing the plasminogen activator with an affinity for the clot. Fears, R., Annals New York Acad. Sci. (1992) 667:343-356; Lijnen, H. R. and Collen, D., Annals New York Acad. Sci. (1992) 667:357-364; and Haber et al., Annals New York Acad. Sci. (1992) 667:365-381. Bi-specific antibodies possessing affinity to both the plasminogen activator and to fibrin have also been constructed for selective accumulation of plasminogen activators in the fibrin clots. Sakharov et al. Thrombosis Res. (1988) 49:481-488. Plasminogen activators have also been conjugated to antibodies specific for collagen for selective delivery of the activator to injured sites of the vascular wall. Muzykantov et al., Biochim. Biophys. Acta (1986) 884:355-363. In addition, monoclonal antibodies recognizing epitopes present on the surface of activated platelets have also been suggested as a targeting vector for thrombolytic agents. Lijnen, H. R. and Collen, D., Annals New York Acad Sci. (1992) 667:357-364. However, the therapeutic potential of such conjugates still remains to be established.
Mutants of plasminogen activators, in particular tPA mutants, have also been constructed which possess altered pharmacokinetic properties and altered functional properties including binding and stimulation by fibrin, and resistance to plasmin and protease inhibitors. Lijnen, H. R. and Collen, D., Annals New York Acad Sci. (1992) 667:357-364. Mutants described to date have had markedly reduced clearances but, usually, also reduced specific thrombolytic potencies.
While several of these approaches are believed to increase thrombolytic potency and prevent rethrombosis, bleeding remains an important issue. None of these approaches provides for selective delivery of the plasminogen activator to the particular tissue where thrombus are most likely to develop, in particular the pulmonary endothelium and the heart.
A multimolecular complex has now been developed comprising a plasminogen activator conjugated to anti-ACE Mab 9B9 which is capable of delivering the plasminogen activator selectively to the pulmonary endothelium. Delivery of plasminogen activators to the surface of the pulmonary endothelium leads to increased concentrations of plasmin in the pulmonary circulation which is useful for enhancing local fibrinolysis in the lungs.